The viral envelope glycoprotein (Env) provides the only known target accessible to neutralizing antibodies. In the virion, the Env exists as a trimeric complex of gp120/gp41 heterodimers that mediates HIV-1 attachment and entry to target cells. To date, all attempts to induce neutralizing antibodies using non-native forms of Env, including monomeric gp120, uncleaved gp160 precursor, and gp41 from which gp120 has dissociated, have failed. While HIV-1 has evolved an Env complex that minimizes the elicitation and binding of neutralizing antibodies, several human monoclonal antibodies have been identified that do potently neutralize primary HIV-1 isolates by recognizing epitopes exposed on the native Env complex. Thus a major challenge in the development of an effective HIV-1 immunogen is to preserve critical aspects of the native, trimeric Env structure in vaccine preparations. Our approach to immunogen design is guided by our recent identification and structural characterization of a novel trimerization domain located within the C-terminal portion of the gp41 ectodomain in the native Env complex. Our preliminary results suggest that an stabilized form of this domain is capable of eliciting broadly neutralizing antibodies. The overall goal of this work is to understand the interaction of neutralizing antibodies with the HIV-1 gp41 trimerization domain and to use this understanding to generate effective immunogens for the elicitation of neutralizing antibodies. Specific aims of this research are: (1) To design and characterize the stabilized forms of the trimeric HIV-1 gp41 membrane proximal domain and to evaluate their immunogenicity in small animals. Our preliminary data indicate that the trimerization domain of gp41 can elicit antibodies that neutralize primary HIV-1 isolates. To confirm and improve upon our initial results, we will construct a carefully selected panel of soluble, stabilized forms of the trimerization domain and evaluate their effectiveness at eliciting broadly neutralizing antibodies in rabbits and mice. (2) To isolate and characterize monoclonal neutralizing antibodies directed against the trimeric HIV-1 gp41 membrane proximal domain and to investigate the mechanistic and structural basis of antibody-trimer interactions. Using both our preliminary constructs and improved constructs generated from Aim 1 as immunogens, we will seek to generate monoclonal antibodies from those animals found to produce neutralizing antibodies. Subsequently, we will study the antigen-binding properties of the neutralizing monoclonal antibodies, in order to clarify the nature of gp41-induced antibody response and specific mechanisms for immune evasion.